Over the past 30 years, medical attention has increasingly turned to the possibility of using proteins as therapeutic drugs for the treatment of disease (See, e.g., “Therapeutic Proteins 1999,” Datamonitor plc, London, 1999). Most protein therapeutics are based on naturally occurring animal or human forms of the protein (such as insulin, growth hormone, erythropoietin, interleukin-2 etc.) and produced in recombinant DNA engineered bacterial or eukaryotic cells. Unfortunately, recombinant proteins made in bacterial cells, such as E. coli, often lack post-translational modifications that are normally found in the protein's naturally produced form. This lack of modification can have a significant negative impact on the pharmaceutical properties of the protein when introduced to a patient.
Glycosylation is one example of a common post-translational modification in eukaryotes that involves the enzymatic attachment of complex sugar chains to the protein. Because recombinant proteins made in bacterial expression systems lack the ability to glycosylate proteins, eukaryotic expression systems such as plant, yeast, insect or mammalian cells have been used in their place when a glycosylated form or ‘glycoprotein’ is required. A significant problem in making recombinant glycoproteins is that the resulting product typically consists of a complex mixture of different ‘glycoforms’, which may have widely varying physiological or pharmacological properties.
Nevertheless, recombinant DNA techniques have become the primary method for commercial production of many polypeptides and proteins because of the large quantities that can be produced in bacteria and other host cells. Recombinant protein production involves transfecting or transforming host cells with DNA encoding the desired exogenous protein and growing the cells under conditions favoring expression of the recombinant protein. E. coli and yeast are favored as hosts because they can be made to produce recombinant proteins at high titers (see, U.S. Pat. No. 5,756,672).
Numerous U.S. patents have been issued with respect to general bacterial expression of recombinant-DNA-encoded proteins (see, for example, U.S. Pat. Nos. 4,565,785; 4,673,641; 4,738,921; 4,795,706; 4,710,473). Unfortunately, the use of recombinant DNA techniques has not been universally successful. Under some conditions, certain heterologous proteins expressed in large quantities from bacterial hosts are precipitated within the cells in dense aggregates, recognized as bright spots visible within the enclosure of the cells under a phase-contrast microscope. These aggregates of precipitated proteins are referred to as “retractile bodies,” and can constitute a significant portion of the total cell protein (Brems et al., Biochemistry (1985)24: 7662.
Recovery of protein from these bodies has presented numerous problems, such as how to separate the protein encased within the cell from the cellular material and proteins harboring it, and how to recover the inclusion body protein in biologically active form. For general review articles on refractile bodies, see Marston, supra; Mitraki and King, Bio/Technology, 7:690 (1989); Marston and Hartley, Methods in Enzymol., 182: 264–276 (1990); Wetzel, “Protein Aggregation In Vivo: Bacterial Inclusion Bodies and Mammalian Amyloid,” in Stability of Protein Pharmaceuticals: In Vivo Pathways of Degradation and Strategies for Protein Stabilization, Ahern and Manning (eds.) (Plenum Press, 1991); and Wetzel, “Enhanced Folding and Stabilization of Proteins by Suppression of Aggregation In Vitro and In Vivo,” in Protein Engineering—A Practical Approach, Rees, A. R. et al. (eds.) (IRL Press at Oxford University Press, Oxford, 1991).
Proteins also have been produced as research reagents by total chemical synthesis (See, e.g., Wilkin et al., Curr. Opinion Biotech. (1999) 9:412–426). This process involves chemical synthesis of proteins by the stepwise coupling of individual amino acids, or convergent strategies that employ the separate synthesis of peptide or polypeptide segments followed by linking of the segments, including chemical ligation of such segments, to generate full-length products (See, e.g., Dawson et al., Ann. Rev. Biochem (2000) 69:923–960). In some instances chemical synthesis has been utilized to make small glycoproteins modified with simple low molecular weight sugars (Shin et al., J. Am. Chem. Soc. (1999) 121:11684–11689). In other cases, proteins have been made chemically to contain detectable labels and the like (Bolin et al., FEBS Letters (1999) 451:125–131). Unfortunately, such labeled proteins offer little to no therapeutic advantage over their recombinantly produced counterparts. However, a few proteins showing therapeutic potential have been made by chemical synthesis with a focus on improving potency by use of small molecule-type changes to pharmacophore regions of the target protein (U.S. Pat. No. 6,168,784). While potency is an important aspect in making a therapeutic, other factors remain problems with protein therapeutics.
Efforts to develop therapeutically usable protein drugs have long suffered from the undesirable bioactivity, bioavailability and biokinetics (such as clearing time, etc.) of putative drug candidates upon in vivo administration. The principal factors that have severely limited the use of proteins, and of polypeptides in particular, as therapeutic agents has been the fact that these compounds often elicit an immunogenic response in the circulatory system (see, U.S. Pat. No. 4,179,337 (Davis et al.)). This effect has one or both of two secondary consequences. The first being the destruction of polypeptides by the elicited antibodies, and the second more seriously, being the emergence of an allergic response. For example, the antibody-mediated destruction of insulin is believed to be responsible for the rather low residence time of insulin in the human circulatory system. In the case of enzymes, not only is there a problem of destruction of the polypeptide and the subsequent negation of its physiological activity but also the most undesired elicitation of an allergic reaction. Conversely, certain proteins, such as clotting factors, growth factors and cytokines, etc., may exhibit a biological half-life that is disadvantageously prolonged upon their in vivo administration.
One approach to improving proteins for use as therapeutics has involved derivatizing the protein with water-soluble polymers. Such conjugation has been proposed as a means for improving the circulating life, water solubility or antigenicity of administered proteins, in vivo (see, U.S. Pat. No. 4,179,337 Davis et al.). Many different water-soluble polymers and attachment chemistries have been used towards this goal, such as polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers) and the like. Polyethylene glycol (“PEG”) is one such chemical moiety that has been used in efforts to obtain therapeutically usable proteins (Zalipsky, S. (Bioconjugate Chemistry (1995) 6:150–165; Mehvar, R. (J. Pharm. Pharmaceut. Sci. (2000) 3(1):125–136)). Its backbone (CH2CH2O)n is flexible, amphiphilic, not susceptible to proteases, and non-immunogenic. Attachment of PEG also has been shown to protect proteins against proteolysis (Blomhoff, H. K. et al., Biochim Biophys Acta (1983) 757:202–208). It also can improve other physical-chemical properties of proteins.
For example, pegademase bovine (Adagen®), a formulation of adenosine deaminase with an attached PEG polymer, has been developed for treating severe combined immunodeficiency disease (SCID); superoxide dismutase with an attached PEG moiety has been in clinical trials for treating head injury; alpha interferon with an attached PEG moiety has been tested for treating hepatitis. Pegylated IL-6 has been described (see, EP 0 442 724, and U.S. Pat. No. 5,264,209). EP 0 154 316 reports reacting a lymphokine with an aldehyde of polyethylene glycol. European Patent Publication EP 0 401 384, describes materials and methods for preparing granulocyte colony stimulating factor (“G-CSF”) to which polyethylene glycol molecules are attached. Modified G-CSF and analogs thereof are also reported in EP 0 473 268, stating the use of various G-CSF and derivatives covalently conjugated to a water-soluble particle polymer, such as polyethylene glycol. U.S. Pat. No. 5,880,255 summarizes many of the proteins that have been modified with PEG chains.
A variety of means have been used to attach polymer moieties such as PEG and related polymers to reactive groups found on the protein (see, U.S. Pat. No. 4,179,337 (Davis et al.), and U.S. Pat. No. 4,002,531 (Royer). For a review, see Abuchowski et al., in “Enzymes as Drugs,” (J. S. Holcerberg and J. Roberts, eds. pp. 367–383 (1981) and Zalipsky, S. (Bioconjugate Chemistry (1995) 6:150–165. The use of PEG and other polymers to modify proteins also is discussed by Cheng, T.-L. et al., Bioconjugate Chem. (1999) 10:520–528; Belcheva, N. et al., Bioconjugate Chem. (1999) 10: 932–937; Bettinger, T. et al., Bioconjugate Chem. (1998) 9:842–846; Huang, S.-Y. et al., Bioconjugate Chem. (1998) 9:612–617; Xu, B. et al. Langmuir (1997) 13:2447–2456; Schwarz, J. B. et al., J. Amer. Chem. Soc. (1999) 121:2662–2673; Reuter, J. D. et al., Bioconjugate Chem. (1999) 10:271–278; Chan, T.-H. et al., J. Org. Chem. (1997) 62:3500–3504. Typical attachment sites in proteins include primary amino groups, such as those on lysine residues or at the N-terminus, thiol groups, such as those on cysteine side-chains, and carboxyl groups, such as those on glutamate or aspartate residues or at the C-terminus. Some of the most common sites of attachment are to the sugar residues of glycoproteins, cysteines or to the N-terminus and lysines of the target proteins.
Although many different approaches have been described for conjugation of polymers to proteins, the conjugation process is not without complications. Care must be taken to limit the loss of biological activity caused by the conjugation reaction. For example, folded or re-folded proteins are typically used to minimize the number of sites of attachment. However, if too much of the activated polymer is attached to the target protein or polypeptide, biological activity can be severely reduced or lost. Likewise, if the wrong linker joining the polymer to the protein is used or an insufficient amount of polymer is attached to the target, the therapeutic value of the resultant conjugate may be limited, and may not demonstrate enough of an increase in circulating life to compensate for the loss in its bioactivity. Problems can also result due to a blockage of the protein's active site by the modifying polymer. This problem can be difficult to avoid since the polymer and protein are typically joined in solution-based reactions. Pre-blocking the active sites with materials such as pyridoxal phosphate has been suggested, but the results have been inconsistent (see, U.S. Pat. No. 4,179,337 (Davis et al.)). These problems are particularly acute with lower molecular weight proteins and peptides, which often have few attachment sites not associated with bioactivity.
For instance, a common technique has been the attachment of water-soluble polymers to the primary amines in the target protein (e.g., the N-terminal amino group and the epsilon amino groups of lysines). Thiol-reactive polymer conjugates also have been used to attach polymers to the thiol side-chains of cysteines. Both approaches represent significant problems as most proteins contain multiple copies of such reactive groups spread out over various regions of the polypeptide backbone that play an important role in defining the activity, folding, re-folding and stability of a protein. Thus although widely used, such approaches suffer from more or less incomplete or unwanted reduction in bioactivity of a protein, and usually complex mixtures that are difficult to separate and characterize (Delgada et al., Pharmaceutical Sciences (1997) 3:59–66).
In an attempt to reduce random attachment, proteins have been made in which the natural lysines are removed in conjunction with adding lysines at the desired sites of polymer attachment (U.S. Pat. No. 4,904,584). For example G-CSF and IL-2 have been modified in this manner. Other attempts to avoid random attachment has been to make proteins in which the natural cysteines are removed in conjunction with adding cysteines at the desired sites of polymer attachment, or cysteines are added without removing the natural cysteines if present (EP 0 668 353). For example, G-CSF, IL-3 and EPO have been made this way. While such cysteine or lysine variants theoretically allow site-specific polymer attachment, there still is no guarantee that all selected sites can be modified in a controlled manner.
Given the inability to site-specifically modify proteins containing multiple amino acids with side-chains bearing the same or similar reactive functional groups, recent efforts have focused on the modification of the amino or carboxy terminus of proteins. Modification of the amino or carboxy terminus has relied on the ability of some chemical conjugation techniques to uniquely modify these sites (WO 90/02136 and WO 90/02135). For example, this technique was utilized for the attachment of PEG chains to the N-terminal residue of G-CSF and the chemokine IL-8 (Gaertner et al., Bioconjug. Chem. (1996) 7(1):38–44; and WO 96/41813). However, modification of the N- or C-termini typically reduces a protein's activity (See, e.g., U.S. Pat. No. 5,985,265 discussing attachment of PEG to the N-terminus and lysine side chains of G-CSF). Despite the drawbacks with modification of proteins with water-soluble polymers, PEGylation and attachment of other water-soluble polymers to proteins continues to be pursued. For example, attachment of PEG to histidine in IF-alpha has been described in U.S. Pat. Nos. 5,951,974 and 6,042,822. Attachment of PEG to lysines in alpha interferon is described in U.S. Pat. No. 5,595,732. Attachment of PEG to sugar chains of erythropoietin (EPO) and internal amino acids such as lysines (EP 0 605 963 and WO 00/32772), and the N-terminus (U.S. Pat. No. 6,077,939 and WO 00/32772) also has been described.
Another problem is that all of the above-discussed polymers are highly non-homogeneous, which render analytical characterization and purification of the mixtures of polymer-modified proteins difficult (Delgada et al., Pharmaceutical Sciences (1997) 3:59–66). For example, techniques used to prepare PEG or PEG-based chains, even those of fairly low relative molecular mass such as 3400, involve a poorly controlled polymerization step which leads to preparations having a spread of chain lengths about a mean value; that is, they involve polymer preparations of (CH2CH2O)n where n does not have a discrete value but rather has a range of values about a mean. The resulting heterogeneity of the derivatized proteins is often associated with a range of properties that one cannot easily identify much less separate. Although the use of polypeptide adducts might be considered to offer a possible solution in principle, the use of polypeptides is disadvantageous since they are susceptible to proteolytic cleavage and can render the derivatized protein immunogenic.
Unfortunately, however, the problem of identifying and employing a suitable polymer is exacerbated by the fact that all proteins currently employed for therapeutic use are derived from recombinant DNA technologies. The use of recombinant DNA technologies puts severe limitations on the types and specificity of linkages that can be formed between a performance-enhancing moiety and the recombinantly produced protein. This is because firstly there are only a very limited number of functional groups suitable for linkage, and secondly there will generally be several copies of the reactive functional group in the protein being modified, thus precluding any specificity of modification. For example, it has been shown that in the case of nonselective conjugation of superoxide dismutase with PEG, several fractions of the modified enzyme were completely inactive (P. McGoff et al., Chem. Pharm. Bull. (1988) 36:3079–3091). Also, if differing numbers of such moieties are randomly attached, the pharmacokinetics of the therapeutic protein cannot be precisely predictable, making dosing a large problem. The lack of control of attachment furthermore may lead to (a) reduced potency, (b) a need for elaborate purification schemes to separate a vast mixture of derivatives and (c) possibly unstable attachment of the modifying moiety. Several linkages, such as tresylchloride-based linkages, known to the art are also known to be immunogenic.
Thus to improve circulating half-life, reduce proteolysis and immunogenicity and improve other properties of biologically produced proteins, water-soluble polymers such as PEG can be attached, but with mixed results given the difficulty of attaching them in a controlled manner and with user-defined precision. Also, because of the limited success in attaching polymers at precise user-defined sites, very little is known about preferred sites of attachment that could be applicable to proteins in general. In addition, because of the stochastic nature of attachment, and the hetero-disperse nature of PEG and other water-soluble polymers currently employed for such purposes, purification and analytical characterization of PEG-protein conjugates has been difficult. Thus the combined problem of poor control over reproducible attachment and polymer heterogeneity has severely hampered the routine approval of polymer-modified proteins as therapeutics (only a few approved to date for therapeutic use despite its introduction in the early 1970's).
Accordingly, a need exists for methods of forming bioactive proteins that are distinct from recombinant DNA technologies, and that could be used to form proteins capable of polymer modification. A need also exists for a preferred polymer that can be employed to form derivatized proteins that have polymer adducts of defined structure rather than a mixture of chains of different lengths. Polymers that can be employed to form derivatized proteins having polymer adducts with structures that can be tailored to mimic desirable properties of natural proteins also is needed. Moreover, a need exists for derivatizing proteins that has general applicability to many proteins. The present invention satisfies these and other needs.